Liver tumor marker sequences

ABSTRACT

Novel polypeptides and nucleic acids that encode the polypeptides are disclosed. The polypeptides and nucleic acids are differentially expressed in liver tumors relative to expression in normal liver tissues. In humans, the nucleic acid sequence maps to a region of chromosome 9p.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/255,674, filed Dec. 14, 2000, which application isincorporated herein by reference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government Support from the followingagency: NIH, Grant No. CA22484. The U.S. Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Liver cancer is the fifth most common cancer worldwide. More than400,000 cases were reported in 1990. Hepatocellular carcinoma (HCC)accounts for 80% of all liver cancer. Liver cancer can result from bothviral infection and chemical exposure. Known risk factors includehepatitis B and C virus infection and exposure to aflatoxin 1. It is notknown whether distinct routes to liver cancer affect the same ordifferent cellular pathways. No mutational model has yet been developedfor liver cancer as it has been for other cancers such as colon cancer.The molecular events that precede neoplastic transformation of the liverare not well understood. With no clearly identified cause, successfultreatment options are lacking. In fact, the specific genes that arederegulated in liver cancer have not yet been enumerated. This is acritical first step in developing a successful strategy for treatingliver cancer.

There is a pressing need to understand the molecular events associatedwith development of liver cancer, both in humans and in animal modelsystems where liver cancer is extensively studied, and to providediagnostic and therapeutic reagents for treating same.

BRIEF SUMMARY OF THE INVENTION

The invention is summarized in that the applicants disclose isolatedpolypeptides whose expression is deregulated in liver tumor cells fromhuman and non-human animals, relative to the expression in regeneratingliver tissue, and further disclose isolated polynucleotides that encodethe isolated polypeptides. As a result of this differential expression,the polypeptides and polynucleotides are diagnostic markers for a livercancer in humans and non-human animals. In humans, the polynucleotidesmap to a region of chromosome 9p.

In one aspect, the polypeptide is selected from the group consisting ofSEQ ID NO:2 and SEQ ID NO:4.

In another aspect, the nucleic acid encodes a polypeptide selected fromthe group consisting of SEQ ID NO:2 and SEQ ID NO:4.

In yet another aspect, the nucleic acid has a nucleotide sequenceselected from the group consisting of an intron-free coding sequencebetween nucleotides 35 and 859 of SEQ ID NO:1 and all of SEQ ID NO:3.The polynucleotides of SEQ ID NO:1 and SEQ ID NO:3 were obtained frommurine and human genetic material, respectively. SEQ ID NO:3 is apredicted spliced cDNA sequence that has been identified in a genomicfragment of the human genome (GenBank Accession No. NT_(—)008335.6,which encompasses sequences previously associated with Accession No.AL391834, named in the above-mentioned provisional patent application).SEQ ID NO:3 is predicted to encode, in humans, a protein within thescope of the invention.

In another aspect, the polypeptide-encoding polynucleotide sequence hasat least about 85% nucleotide sequence identity to the coding sequenceof SEQ ID NO:1 or SEQ ID NO:3 (using the NCBI Blast 2 comparisonprotocol) where the polynucleotide hybridizes under stringenthybridization conditions to the polynucleotide of SEQ ID NO:1 or SEQ IDNO:3. Also within the scope of the invention is a nucleic acid having atleast about 90%, and most preferably at least 95% identity to eithersequence.

In a related aspect, a polynucleotide sequence having greater than 90%homology to the protein-encoding sequences of SEQ ID NO:1 has beenidentified in a region of human chromosome 9p. A putative proteinencoded at that location is greater than 90% similar to the amino acidsequence of SEQ ID NO:2.

In another aspect, the nucleic acid hybridizes under moderatelystringent hybridization conditions to a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:3.

In a related aspect, the nucleic acid hybridizes under highly stringenthybridization conditions to a nucleotide sequence selected from thegroup consisting of SEQ ID NO:1 and SEQ ID NO:3.

In another related aspect, the invention is an oligonucleotide thathybridizes under highly stringent or moderately stringent conditions toa polynucleotide of the invention.

In another aspect, a polynucleotide of the invention, whether from miceor humans or any other source, is engineered into a genetic constructdownstream from a heterologous promoter not natively upstream of thepolynucleotide that directs expression of the encoded protein. Thegenetic construct is introduced into a host cell that supportstranscription of the polynucleotide and translation of the protein whichcan then be purified using methods known to those skilled in the art.Alternatively, the construct can be provided in an in vitrotranscription/translation system for protein production.

In still another aspect, the invention is an antibody that specificallybinds to a polypeptide of the invention.

In yet another aspect, the invention is a method for identifyingmodulators (inducers or suppressors) of expression of thepolynucleotides and polypeptides of the invention, where the methodincludes the step of observing a change in level of expression of apolynucleotide or polypeptide of the invention in a host cell thatexpresses the polynucleotide or polypeptide after exposure of the hostcell to a modulating agent.

It is an object of the present invention to provide an isolated nucleicacid and an isolated polypeptide that are associated with hepatocellularcarcinoma in human and non-human animals.

In yet another aspect, the present invention provides a host celltransfected with the genetic construct described above.

In still another aspect, the invention can relate to a kit having use ina method for determining in a tumor or other cell the expression levelof the polypeptide or of a nucleic acid encoding the polypeptide. Thekit can contain one or more antibody directed to an epitope on thepolypeptide and one or more oligonucleotide or polynucleotide thathybridizes to the nucleic acid that encodes the polypeptide. The kit canalso further include additional components for use as positive ornegative controls in a method for determining the expression level. Suchadditional components can include samples of tumor or non-tumor livercells, or an extract of any of the foregoing, for which a level ofexpression of a polypeptide or a polynucleotide of the invention hasbeen determined. Alternatively or additionally, the kit can contain asample of one or more of a polypeptide, a polynucleotide, and anoligonucleotide of the invention for quantification purposes.

Other objects, features and advantages of the present invention willbecome apparent upon consideration of the following detailed descriptionof the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

Liver cancer is generally studied in animal model systems, preferably inrodent systems, where certain strains have been bred for their highsusceptibility to liver tumors. C3H/HeJ mice are highly susceptible toliver tumors after induction with diethylnitrosamine (DEN). To identifypolynucleotide sequences or genes that show differential expression inliver tumor cells as compared to normal liver tissue cells, geneexpression differences between liver tumors and a regenerating liverwere determined using representational difference analysis (RDA:Lisitsyn, et al., Science 259:946 (1993), incorporated by reference asif set forth herein in its entirety.

In this application, the applicants report the amino acid sequences of apair of polypeptides from murine animals and humans (and the sequencesof the nucleic acids that encode the polypeptide sequences) that arehighly differentially expressed in cells of human and non-human livertumors relative to regenerating normal liver cells. The polypeptide isconveniently referred to as CRG-L1 although the designation is merelyarbitrary. Further, the invention provides materials and methods fordetecting expression (and changes in expression) of the nucleic acids(including mRNA, single or double stranded DNA, cDNA and the like) andproduction of the polypeptides, thereby facilitating use as a diagnosticmarker for liver cancer and as a system for assessing putativetherapeutic agents.

The polypeptides and nucleic acids of the invention can be isolated andpurified from normally associated material in conventional ways suchthat in the purified preparation the polypeptide or nucleic acid is thepredominant species in the preparation. At the very least, the degree ofpurification is such that the extraneous material in the preparationdoes not interfere with use of the polypeptide or nucleic acid of theinvention in the manner disclosed herein. The polypeptide or nucleicacid is preferably at least about 85% pure, more preferably at leastabout 95% pure and most preferably at least about 99% pure.

Structurally, the nucleic acid sequence of murine CRG-L1 (SEQ ID NO:1)encodes a polypeptide of about 275 amino acids with a predictedmolecular weight of about 30 to 35 kDA. In particular, the murine CRG-L1includes seven putative transmembrane domains that correspond to aminoacids 33–53, 62–82, 91–111, 123–143, 146–166, 174–194, and 212–232 ofSEQ ID NO:2.

The nucleic acid sequences of the invention can be introducedconventionally into, and expressed in, host cells which can beprokaryotic (such as bacteria) or eukaryotic (such as yeast, insect,amphibian or mammalian cells) whereupon the transcription of nucleicacid and the properties of the encoded proteins can be assessed.

The isolation of a biologically active polypeptide that isdifferentially regulated in a liver tumor provides a means for assayingfor inhibitors and activators (in vivo or in vitro) of such apolypeptide that can affect the development or progression of livertumors. For example, the polypeptide can be expressed in cells and theeffect of various test agents on mRNA or protein expression levelrelative to untreated controls can be measured. Alternatively, the levelof expression can be assessed in biological samples taken directly froma human or non-human tissue.

The presence and level of such a differentially regulated protein can bereadily discerned using antibodies directed to an epitope on the proteinusing well known methods, such as an ELISA method. The level of geneexpression in a liver tumor and in regenerating liver tissue can also bemeasured using methods for hybridizing nucleic acids (including, withoutlimitation, RNA, DNA, and cDNA). Such methods are generally known tothose skilled in the art, but are enabled by the disclosure herein of aliver tumor-specific sequence. Because one can assess levels ofexpression of protein and nucleic acid, it is also, therefore, possibleto develop agonists and antagonists of the encoded protein or toidentify agents that affect transcription or translation of thedisclosed nucleic acid sequences.

A skilled artisan understands that polypeptide sequences presentedherein can vary somewhat, whether as a result, e.g., of sequencing erroror allelic variation or duplication, from the sequence presented whilestill retaining their essential nature, that is, differential regulationin liver tumors relative to normal liver tissue. Further, the nucleicacids of the invention include conservatively modified variants of thesequences presented herein, complementary sequences, and splicevariants. In view of the known degeneracy in the genetic code, theproteins disclosed can also be encoded by a large number of otherpolynucleotide sequences, all of which are within the scope of theinvention. The polypeptides of the invention includes polymorphicvariants, alleles, mutants, and interspecies homologs that (1) aredifferentially expressed in liver tumors, (2) bind to antibodies raisedagainst the coding region of either disclosed polypeptide, (3)specifically hybridize under stringent hybridization conditions to anucleic acid sequence selected from a group consisting of SEQ ID NO:1and SEQ ID NO:3, or (4) are amplified by primers that amplify SEQ IDNO:1 and SEQ ID NO:3.

Exemplary high stringency hybridization conditions include 50%formamide, 5X SSC and 1% SDS incubated at 42° C., or 5X SSC and 1% SDSincubated at 65° C., followed by washing in 0.2X SSC and 0.1% SDS at 65°C. Exemplary moderate stringency hybridization conditions include 40%formamide, 1M NaCl and 1% SDS incubated at 37° C. followed by washing in1X SSC at 45° C. These conditions are merely exemplary as one skilled inthe art is readily able to discern stringent from moderately stringenthybridization conditions.

Moreover, the sequences of the invention also encompass substitutions,additions and deletions of the sequences presented where the changeaffects one or a few amino acids in the presented polypeptide sequences,without substantial effect upon the activity of the polypeptide.

The present invention will be better understood upon consideration ofthe following non-limiting example.

EXAMPLE

Inbred C3H/HeJ mice were bred and housed in plastic cages on corncobbedding (Bed-O'Cobs; Anderson Cob Division) and were fed Breeder Blox(Harlan). Food and acidified water were available ad libitum. To obtainregenerating livers, partial hepatectomies were preformed on male, sixweek old mice as described by Lukas, E. R., et al., MolecularCarcinogenesis 25:295–303 (1999). All papers mentioned in the exampleare incorporated by reference herein as if set forth in their entirety.Animals were sacrificed 36 hours after the surgery, at a time thatcorresponds to peak DNA synthesis, and the liver remnants wereharvested.

Liver tumors were taken from male C3H/HeJ mice that had been treatedwith DEN (0.1 μM/g of body weight) at 12 days of age and sacrificed at32 weeks of age.

Total RNA was extracted from liver using guanidine thiocyanate/CsCl asdescribed by Lukas et al. PolyA mRNA was isolated from 250 μg of totalRNA using Oligotex mRNA kit (Qiagen). The cDNA RDA protocol developed byHubank, M. and D. G. Schatz, Nucleic Acid Research 22:5640–5648 (1994)was followed in detail using polyA RNA from the regenerating livers andthe liver tumors. cDNA RDA is a method for cloning transcripts found inthe pool of mRNA from one source, but absent from the pool of mRNA froma second source. Depending upon how the experiment is set up, one canidentify novel genes that are either up-regulated or down-regulated.Briefly, mRNA is obtained from two tissues, in this case regeneratinglivers and liver tumors. The cDNA RDA technique is performed on cDNAprepared using standard methods from the isolated mRNA.

In the first subtractive round, the representations were hybridized toeach other in a 1:100 tester/driver ratio. The second and thirddifference products used a tester/driver ratio of 1:800 and 1:400,000respectively. Difference products were subcloned from the seconddifference product subtractive round because no products were observedin the third round. Cloned products were sequenced by Big Dye Sequencing(Applied Biosystems, Inc.). Two comparisons were performed between theregenerating livers and the liver tumors. In the first comparison, thetester, provided in a more limited amount than the driver, was cDNAderived from the liver tumor. This comparison can identify genesupregulated in liver tumors. In the second comparison, the tester wascDNA derived from the regenerating liver. This comparison can identifygenes upregulated in regenerating livers. A number of differenceproducts were obtained in each comparison, indicating that some mRNAsare up-regulated in liver tumors while other mRNAs are down-regulated inliver tumors, relative to regenerating liver tissue. Many of theup-regulated and down-regulated sequences correspond to known genes. Inaddition, however, five novel differentially expressed transcripts werealso identified.

The most highly differentially expressed polynucleotide sequence wasexamined for expression in eight mouse tissues and in four embryonictissues using a multiple tissue cDNA panel (Clontech) according to themanufacturer's recommended protocol. The isolated RDA fragment includedbases 997 through 1383 of SEQ ID NO:1. The polynucleotide was expressedmost highly in heart, lung, and testes. Modest expression was seen inregenerating liver, which is consistent with the low levels observed inthe RDA when compared to a liver tumor.

A cDNA clone of the differentially expressed polynucleotide was obtainedby screening the Origene rapid-screen mouse liver cDNA library withprimers designed from the isolated RDA fragment. A 4.175 kb cDNA wasisolated and sequenced. At the 3′ end of the cDNA, significant homologyto twenty-three mouse ESTs (GenBank Accession Numbers AA048715,AA212916, AA212925, AA462019, AA462654, AA475320, AA914194, AV227941,AW490555, AW701866, AW702104, BB108761, BB627599, BB660847, BB752973,BB764116, BF144307, BF468547, BF661433, BF662488, BG109928, BG230006,and BI080821) was noted. An ATG translation initiation site was seen atbase pairs 35–45 of SEQ ID NO:1, with an open reading frame extending tobase 862, and followed by a stop codon. The predicted translationproduct is a protein of 275 amino acids having a molecular weight of31.4 kD. Seven putative transmembrane domains were revealed using theSMART (Simple Modular Architecture Research Tool) which analyzes proteinsequences for motifs. The transmembrane domains correspond to aminoacids 33–53, 62–82, 91–111, 123–143, 146–166, 174–194, and 212–232 ofSEQ ID NO:2. The existence of related sequences in C. elegans and D.melanogaster suggest a conserved function for the polynucleotideobtained by the inventors.

In the human genome data base at NCBI, clone Hs9_(—)8492 (GenbankAccession No. NT_(—)008335.6, a contig from human chromosome 9p, wasshown to have areas of significant homology to the entire mouse cDNAsequence. In this clone obtained from human DNA, six exons havingsignificant homology to the mouse polynucleotide sequence wereidentified. The entire sequence in humans of an open reading frame thatcorresponds to murine SEQ ID NO:1 is represented in clone Hs9_(—)8492.

The human sequences thus identified were joined with reference to thedisclosed SEQ ID NO:1 by removing putative splice regions and pastingthe remaining sequences together to join as SEQ ID NO:3 the followingareas of the contig, in this order: 660483–660351, 645683–645565,644843–644705, 634599–634459, 623266–623125, and 619093–618907 (as thosesequences are numbered as of the filing date of the application in thesequence of Hs9_(—)8492, which contains a set of 21 as yet unlocalizedpieces. This single clone includes all of the sequences that, whenarranged to form coding sequence 1–825 (plus a stop codon) of SEQ IDNO:3, correspond to a polynucleotide sequence from bases 35 to 862 ofSEQ ID NO:1 from mice. Further, such a sequence can encode a protein inhumans that corresponds to the protein of SEQ ID NO:2. The putativehuman cDNA is 87% identical to the mouse sequence. If the putative humancDNA is translated, the resulting amino acid sequence is 91% similar tothe corresponding portion of the mouse amino acid sequence, using theLipman-Pearson protein alignment with a gap penalty of 4 and gap lengthpenalty of 12.

Sequences corresponding to the basal promoter region have also beenidentified within Hs9_(—)8492 from 661112 to 660393. SPI (660696–660703and 660616–660622) and E2F (660543–660551) transcription factor bindingsites have also been identified upstream of the coding sequence of thehuman coding sequence. Cotransfections using luciferase reporters haveshown that the CRG-L1 promoter is activated by E2F1.

The polynucleotide and polypeptide sequences provide a skilled artisanwith the ability to assess using conventional methods the expressionlevels of this human gene and array of tissues and more specifically tomonitor the expression of the gene in human liver tumors as compared tonormal human liver tissue. Likewise, antibodies directed to a portion ofthe human protein can be produced and used as diagnostic agents forassessing protein levels in various human tissues including livertumors.

The applicants have observed by RT-PCR analysis that, like the murinemRNA, the human CRG-L1 mRNA is upregulated relative to normal livertissue in three different surgically-excised human hepatocellularcarcinomas and one hepatocellular adenoma. The mRNA level in thesesamples was comparable to that observed in the HepG2 hepatocellularcarcinoma cell line. Differential expression of CRG-L1 mRNA was notobserved in human colon adenocarcinomas.

The applicants have also observed that other proteins from mouse andhuman libraries interact with the C-terminal domain in a yeasttwo-hybrid screen, namely clathrin adapter protein AP-1, megakaryocytestimulating factor and Jab-1.

The present invention is not intended to be limited to the foregoing,but rather to encompass all such variations and modifications as comewithin the scope of the appended claims.

1. An isolated nucleic acid comprising a coding sequence for apolypeptide selected from the group consisting of SEQ ID NO:2 and SEQ IDNO:4.
 2. A genetic construct comprising a polynucleotide of claim 1downstream from a heterologous promoter.
 3. A host cell transfected withthe genetic construct of claim
 2. 4. A method for identifying modulatorsof expression of a polynucleotide consisting of a coding sequence for apolypeptide selected from the group consisting of SEQ ID NO:2 and SEQ IDNO:4, the method including the step of observing a change in the levelof expression of the polynucleotide in the host cell of claim 3 afterexposure of the host cell to a modulating agent.
 5. The isolated nucleicacid of claim 1, wherein the coding sequence is selected from the groupconsisting of the coding sequence of SEQ ID NO:1 and the coding sequenceof SEQ ID NO:3.